SB 741 
.L4 07 

'°''^ ' LEAF SMUT OF TIMOTHY 



A THESIS 

Presented to the Faculty of the Graduate School 
OF Cornell University for the degree of 

DOCTOR OF PHILOSOPHY 



BY 

GEORGE ADIN OSNER 



Also published as Bulletin 381 of the Cornell University Agricultural Experiment 
Station, October, 1916. 



LEAF SMUT OF TIMOTHY 



A THESIS 

Presented to the Faculty of the Graduate School 
OF Cornell University for the degree of 

DOCTOR OF PHILOSOPHY 



BY 

GEORGE ADIN OSNER 



Also published as Bulletin 381 of the Cornell University Agricultural Experiment 
Station, October, 19 16. 



SB 7-?/ 



la foehang*. 
DEC 2 3 19ie 






CONTENTS 

PAGE 

Hosts 1 89 

The disease 1 90 

Names 190 

History and distribution 1 90 

Economic importance 1 90 

Symptoms 194 

On the leaves 194 

On the inflorescence 199 

Etiology 199 

History and classification of the pathogene 199 

Morphological and life history studies 201 

Spores 201 

Morphology 201 

Germination 203, 

Mycelium 209 

Spore formation 212 

Inoculation and infection 216 

Seed inoculation 217 

Blossom inoculation 21S 

Soil inoculation 219 

Inoculation of growing tissues 219 

Examination of inoculated seedlings 220 

Summary of life history 220 

Pathological histology 221 

Effect of environmental factors 222 

Control 223 

Effect of seed treatment on germination 223 

Effect of seed treatment on percentage of smut 225 

Bibliography 226 



187 



LEAF SMUT OF TIMOTHY ^ 

George A. Osner 

HOSTS 

Leaf smut of timothy has been reported on a large number of grasses 
of the subfamily Poacoideae, of the Gramineae. To give an accurate 
and complete list of all the hosts affected by this disease will not be 
possible until the morphological and biological limits of the causal organism 
shall have been determined by careful comparison and cross-inoculation 
on the various European and American hosts. The following list con- 
tains the more important hosts mentioned as subject to the disease, but 
the Hst is not claimed to be complete: 

Agrostis alba L. (redtop), Agrostis alba var. vulgaris (With.) Thurb., 
Agrostis stolonifera L. (creeping bent), Alopecurus pratensis L. (meadow 
foxtail), Ammophila arenaria (L.) Link (beach grass), Anthoxanthum 
odoratum L. (perennial sweet vernal grass), Arrhenatherum elatius (L.) 
Beauv. (tall oat grass), Avena pubescens Huds., Brachypodium pinnatuni 
Beauv., Brachypodium sylvaticum Beauv., Briza media L. (perennial 
quaking grass), Bromus erectus Huds., Bromus inermis Leyss. (Hungarian 
brome grass), Dactylis glomerata L. (orchard grass, or cocksfoot), 
Deschampsia caespitosa (L.) Beauv., Elymus canadensis var. glaucifolius 
(Muhl.) Gray (glaucous wild rye), Elymus glaucus Buck, (smooth wild 
rye), Elymus robustus Scr. & J. G. Sm., Elymus virginicus L. (Virginia 
wild rye), Festuca distans Kunth, Festuca elatior L. (meadow fescue), 
Festuca nutans Spreng. (nodding fescue), Festuca ovina L. (sheep's fescue), 
Festuca ovina var. duriuscula (L.) Hack., Festuca ovina var. glauca Hack., 
Holcus lanatus L. (velvet grass), Holcus mollis L., Lolium multiflorum 
Lam. (awned, or Italian, rye grass), Lolium perenne L. (ray grass), Milium 
effusum L. (millet grass), Phleum pratense L. (timothy), Poa annua L. 
(low spear grass), Poa bulbosa L., Poa debilis Torr. (weak spear grass), 
Poa nemoralis L., Poa pratensis L. (Kentucky bluegrass), Poa trivialis 
L. (rough-stalked meadow grass), Sitanion longifolium J. G. Sm. (long- 
bristled wild rye). 

The writer has observed this disease on the following plants in New 
York: Agrostis alba var. vulgaris (With.) Thurb., Agrostis alba var. unde- 
termined (a creeping variety), Dactylis glomerata L., Phleum pratense 
L., Poa annua L.. and Poa pratensis L. 



' Also presented to the Faculty of the Graduate School of Cornell University, June, IQIS, as a major 
thesis in partial fulfillment of the requirements for the degree of doctor of philosophy. 

Author's acknowledgments. The writer wishes to acknowledge his indebtedness to Professors 
Donald Reddick and H. H. Whetzel, of the Department of Plant Pathology, Cornell University, for helpful 
suggestions and criticisms during these investigations, and to H. H. Knight for a number of the photo- 
graphs that are here reproduced. 

189 



igo Bulletin 38 i 

The only mention in literature of varietal susceptibility is by Clinton 
(1900);- who states that the fungus causing leaf smut is most injurious 
to redtop. The writer has found this true for New York. He has never 
found Canada bluegrass infected, altho it frequently occurs in association 
with diseased Kentucky bluegrass. 

THE DISEASE 

NAMES 

The term leaf smut of timothy, which is employed by the writer to 
designate this disease, was first used in this country by Trelease (1887). 
The name is not entirely applicable, since the lesions are by no means 
limited to the leaves. However, since the lesions on the leaves constitute 
the most characteristic symptom, this name is retained. In Denmark 
the name graessernes stinkhrand (stinking smut of grasses) has been used, 
probably on the supposition that the pathogene is closely related to that 
of the stinking smut of wheat (Rostrup, 1904). 

HISTORY AND DISTRIBUTION 

The origin of this disease is unknown. It was first recorded from 
Italy on Holcus mollis by Cesati (1850). Westendorp (1852) records it 
from Belgium on velvet grass {Holcus lanatus) and perennial sweet vernal 
grass {Anthoxanthum odoratiim). It has since been reported from various 
European countries and from Australia as being more or less common. 

The first mention of the disease in North America was by Trelease 
(1885 a), in a paper read before the Wisconsin Academy of Science in 
December, 1882. He records it from Wisconsin on timothy (Phleum 
pratense) and on glaucous wild rye {Elymus canadensis var. glaucif alius) . 
Trelease (1885 b) also published the first economic account of leaf smut, 
stating that it had been very prevalent in Wisconsin for the previous 
two seasons. Clinton (1906) gives its present distribution in North 
America as: California, Connecticut, Delaware, District of Columbia, 
Illinois, Indiana, Iowa, Kansas, Maine, Massachusetts, Minnesota, 
Missouri, New Jersey, New York, Ohio, Texas, Utah, Washington, Wis- 
consin, and Canada. The writer has observed it in several counties of 
New York and Indiana. 

ECONOMIC IMPORTANCE 

Economic loss from this disease occurs in two ways. First, thru a 
reduction in the yield of hay, and second, thru a reduction in the yield 

2 Dates in parenthesis refer to bibliography, page 226. 



Leaf Smut of Timothy 



191 



of seed. The fact that diseased plants are usually stunted in growth 
is probably the reason why the disease is so generally overlooked. Even 




Fig. 45. LEAF SMUT ON TIMOTHY 

Healthy plant (at left) contrasted with diseased, stunted plants 

in badly infested fields the grower is likely to attribute the reduction in 
yield to the weather or to other external factors. 



192^ 



Bulletin 381 



Several writers have reported leaf smut as causing considerable damage 
to meadows. Clinton (1900) says that in 1898 he found a field of redtop 
injured thirty per cent. The owner stated that at times the injury had 
cut down the yield of seed from the normal 300 hundredweight to 70 
hundredweight. Pammel (1892 a) reports considerable loss on the Iowa 




Fig. 46. TIMOTHY PLANTS KILLED BY THE LEAF SMUT FUNGUS 

College farm from the disease. He states (1893) that it can be found in 
most timothy fields. Trelease (1885 b) says the fungus caused consider- 
able loss about Madison, Wisconsin, in 1883 and 1884. Griffiths (1903) 
reports damage to timothy in Jess Valley, California. 

Leaf smut is extremely common in New York. In the summer of 
1 9 14 the writer examined a large nimiber of timothy fields in nine counties, 



Leaf Smut of Timothy 



193 



and found the disease in more or less abundance in every field, in one 
field over fifty per cent of the stools were aftected. The loss of hay in 




Fig. 47. LESIONS of leaf smut on leaves and inflorescence of timothy 

Leaves show the typical tearing, or shredding 

this field was estimated to be about thirty per cent. If the timothy had 
been grown for seed the loss would have been greater. In 19 14 the 



194 



Bulletin 381 



disease caused a reduction in the yield of hay in Genesee County of 
probably not far from four per cent. In other counties the writer has 
not examined a sufficient number of fields to be able to speak with certainty 
as to average losses. 

SYMPTOMS 

The diseased plants are usually more or less stunted (fig. 45)- They 
may be found showing all degrees of this dwarfing, from plants not over 




Fig. 48. soRi of ustilago striaeformis in leaves of timothy 

One plant shows sori in the young unfolded leaf. The chlorophyll had been 
partly removed before taking the photograph. A, natural size; B, X 3 

four or five inches high and with only three or four leaves to those that 
are apparently equal in vigor to the healthy plants. Frequently the 
more diseased culms in a stool are much dwarfed, while the others are 
nearly normal. Later in the summer some of the smaller plants will be 
found to have been killed outright (fig. 46). 

On the Leaves 

The disease shows first as elongate, narrow striee on leaves and sheaths, 
and later appears on the stems (figs. 45, 47, and 48). In the latitude of 



Leaf Smut of Timothy 195 

New York the sori do not become conspicuous until about the first of 
May, but on careful search they may be found any time during the winter 
on plants that have not been killed back entirely by frost. When first 
visible they may be not over one-tenth of a millimeter in width and two- 
tenths of a millimeter in length, but are usually from two-tenths to four- 
tenths of a millimeter in width by from one-half to one millimeter or 
more in length. Later, by fusion of the sori end to end, they may become 
several centimeters long or may even extend thruout the length of the 
leaf and down the sheath. Occasionally the sori may also fuse later- 
ally. The number of sori on a leaf may vary from one to several, in some 
cases nearly the whole surface of the leaf being covered. 

At first the sorus may be visible on only one surface, depending on 
whether it originates nearer the upper or the lower epidermis. Later it 
usually extends thru the leaf from surface to surface, being covered only 
by the epidermis, which gives it a lead-colored appearance. As the 
spores mature, the sorus increases in size, pushing up the epidermis one- 
tenth of a millimeter or more (Plate xvii, 6). Later the epidermis rup- 
tures, exposing the dark brown or nearly black, dusty mass of spores 
beneath. These spores are scattered by the wind and the leaves become 
very much torn and shredded (fig. 47). This shredded appearance of 
the leaves is one of the most striking symptoms on the older plants, enabling 
one to recognize the disease at a considerable distance. As the leaves push 
out at the tip of the growing plant, the lead-colored sori are often found 
already present (fig. 48, a), and in badly diseased plants these sori may 
extend down to the base of the stem. If the stem is cut across a short 
distance back of the growing tip, the black spore masses may be found 
in the outer cortex (Plate xvii, 4). 

There is usually little or no difference in color between diseased and 
healthy plants, unless the leaves become so badly diseased that the 
tissues between the sori die; in such cases the leaves become yellow or 
brownish. 

The symptoms of the disease on the leaves of other grasses observed 
are very similar to those on timothy. On redtop, however, the tendency 
to form sori extending thruout the length of the leaf and down the 
sheath is much more pronounced than on timothy. The most striking 
characteristic of the disease on redtop is the tendency of the leaves at 
the top to become badly shredded (fig. 49). Its dwarfing effect on Ken- 
tucky bluegrass and on orchard grass is well shown in figures 50 and 51, 
respectively. In the case of Kentucky bluegrass, especially, the diseased 
plants are very easily overlooked because of their small size. 



ig6 



Bulletin 381 




Fig. 49. LEAF SMUT ON REDTOP, SHOWING LEAVES AT THE TOP BADLY SHREDDED 



Leaf Smut of Timothy 



197 




Fig. 50. LEAF SMUT ON KENTUCKY BLUEGRASS 

Healthy plant (at right) contrasted with diseased, stunted plant 



1 98 



Bulletin 381 




Fig. 51. LEAF SMUT ON ORCHARD GRASS 

Healthy plant at right. (Photograph taken in the field) 



Leaf Smut of Timothy 



199 



On the inflorescence 

Usually the diseased plants do not fruit. On those that do, the sori 
appear at an early stage as more or less elongated striae on the rhachis 
or in the florets (fig. 47). In the 
florets any or all of the parts may 
be broken down and replaced by 
the spore mass (fig. 52). In severe 
attacks all parts, even including the 
bristles, may be destroyed. The sorus 
may be produced either before or 
after the glumes have attained nearly 
full growth, and in the latter case 
usually only a part of the glume is 
destroyed. 

The inflorescence of redtop is 
usually diseased at the time it emerges 
from its sheath, and only rarely do 
diseased plants produce viable 
seed. Of the various hosts observed, 
viable seed is produced on diseased 
orchard grass oftener than on any 
other. 




Fig. 52. HEALTHY AND DISEASED TIMOTHY 
SEED AND GLUMES 

Healthy seed in top row at right. The seed in 
the smutted glumes has been destroyed. All taken 
from the same inflorescence. X 7 



ETIOLOGY 

History and classification of the pathogene 

The organism causing leaf smut has been collected and described, 
under a number of different names, by various investigators. This 
is due in large measure to the fact that it occurs on such a wide range 
of host plants. It was first collected by Cesati on Holcus mollis and 
distributed in Klotzsch-Rabenhorst's Herbarium Vivum Mycologicum 
(1850) as Uredo longissima var. Hold. Westendorp (1852) described it 
from Holcus lanatus as a new species, giving it the name Uredo striaeformis , 
probably adopting this name because of the characteristic appearance 
of the lesions on leaves and stem. Fischer von Waldheim (1866) described 
this fungus from Holcus mollis as Tilletia de Bar y ana. He placed it in 
the genus Tilletia largely on the basis of its method of spore formation, 
which he reported to be on the ends of side branches. Most European 
mycologists have since followed this worker, placing the fungus in the 
genus Tilletia. Oudemans (1878) pointed out that, adopting the first 
specific name applied to the organism, it should be called Tilletia striae- 
formis. Niessl (1876), believing that the fungus was a species of 
Ustilago rather than of Tilletia, stated that it should be called Ustilago 



Plate XVII. photomicrographs of mycelium and sori of ustilago 

STRIAEFORMIS 

1, Cross section through base of a timothy stem, showing intracellular mycelium. X 700 

2, Cross section through base of a timothy stem, showing mycelium in a vascular bundle. 
X315 

3, Part of a sorus from a stem of Daclylis glomerala, showing large spores in the center, 
and smaller, less mature ones at the edge. X s6o 

4, Cross section of timothy stem, showing two sori in the outer cortex just beneath the 
epidermis. The ring of heavy-walled sclerenchyma cells containing isolated strands of myce- 
lium is shown just inside these sori. X 75 

5, Part of redtop leaf showing sori of various ages between the vascular bundles. Some 
of the sori are in process of fusing. X 65 

6, Cross section of timothy leaf, showing two sori. The epidermal cells are hyper- 
trophied and considerably bulged. X 75 



BlLLETIN 3S1 



Plate XVII 



■nr 









PHOTOMICROGRAPHS OF MYCELIUM AND SORI OF USTILAGO STRIAEFORMIS 



Leaf Smut of Timothy 26f 

striaeformis (West.)- Sporidia of this fungus, with the exception of a 
rather unsatisfactory figure by Pammel, Weems, and Lamson-Scribner 
(1901), have never been described ; consequently the generic name can be 
determined only indirectly. The writer has adopted the name Ustilago 
striaeformis (West.) Niessl, basing his decision on the method of spore 
formation and spore germination as stated elsewhere (page 209). 

A number of closely related species have been described, some of which 
may eventually prove to be identical with this fungus. Among those that 
apparently are distinct may be mentioned Ustilago Salveii Berk. & Br., 
Ustilago macrospora Desm., and Ustilago Calamagrostidis (Fckl.) Clinton. 

A list of the more important names applied to this fungus is as follows : 

Uredo longissima var. Hold Ces. 

Klotz.-Raben. Herb. viv. mycol., no. 1498. 1850. 
Uredo striaeformis West. 

Acad. Roy. Belgique. Bui. 18, scr. 2:406. 1852. 
Uredo longissima var. megalospora Riess 

Klotz.-Raben. Herb. viv. mycol., no. 1897. 1854. 
Tilletia de Baryana F. de W. 

Raben. Fungi eur., no. 1097. 1866. 
Tilletia Milii Fckl. 

Symb. myc. 1:40. 1869. 
Ustilago striaeformis (West.) 

Niessl in Hedwigia 15: i. 1876. 
Tilletia striaeformis 

Oudemans in Bot. Ztg. 36:440. 1878. 
Tilletia striaeformis (Westd.) 

Winter in Krypt. -Flora. Pilzeii:io8. 1880. 
Tilletia alopecurivora Ule 

Bot. Ver. Prov. Brandenburg. Verb. 25:214. 1884. 
Tilletia Brizae Ule 

Bot. Ver. Prov. Brandenburg. Verb. 25:214. 1884. 
Tilletia striiformis (Westend.) Magnus 

Saccardo in Syll. fung. 7-: 484. 1888. 
Ustilago poarum McAlp. 

Roy. Soc. Victoria. Proc. n. ser. 7:220. 1894. 
Ustilago Washingtoniana Ell. & Ev. 

Eul." Torr. Bot. Club 22 : 57. 1 895. 
Tilletia airae-cespitosae Lindr. 

Soc. pro Fauna et Flora Fennica. Acta 26: 15. 1904. 

Morphological and life history studies 
Spores ^ 

Morphology. — The spores of this fungus vary from spherical to ellipsoidal 
or irregular. In sori in which the spores are not greatly crowded most 
of them are nearly spherical, while in sori in which much pressure has 
occurred the spores are found to be very irregular in shape (Plate xvii, 3). 
In mass they are nearly black, but as seen under the microscope they are 
olive-brown in color. 

The spores measure from 10 to 17M by from 8 to i2ju; but out of several 
himdred spores measured from the various hosts observed, the writer 

' The term spore is used thruout this paper in preference to the word chlamydospore. 



202 



Bulletin 381 



has found the majority to fall within the limits 10 to 14M by 8.5 to ii^. 
The spore is covered with a thick wall, which is divided into two layers — 
a hyaline inner endospore and a darker, thicker exospore (fig. 53, e). 
The latter varies from echinulate to verrucosa even in viable spores from 
the same plant. These spines or warts are usually rather blunt, and 




Fig. 53. SPORE formation of ustilago striaeformis 

A, Immature spores from Dactylis glomerata, showing various stages in the fusion of the nuclei. In 
two spores the nuclei are not yet fused. One spore has a single nucleus with the nucleoli not yet fused. 

X 1670 

B, C, Various stages in the maturation of spores from Dactylis glomerata. X 1670 

D, Mature spores from Dactylis glomerata. X 1670 

E, Mature spore from timothy, showing endospore and vacuoles, or oil globules. X 3530 



on mature spores (fig. 53, d, e) are about one micron in length. They 
may be close together or may stand a considerable distance apart. The 
endospore is difficult to discern in fresh spores, but becomes more readily 
apparent if the spore is held for a few minutes in dilute sulfuric acid. 
The spores contain large oil globules, which are usually more readily seen 



Leaf Smut of Timothy 203 

after treatment with dilute potassium hydroxide solution. The mature 
spores each have a single nucleus, varying from 2.5 to 5.5^1 in diameter. 
Each nucleus has a single large nucleolus. 

Germination.'^ — In literature only a few investigators have reported 
germination of the spores of this fungus. Pammel (1893) says the spores 
germinate readily. Pammel, Weems, and Lamson-Scribner (1901) report 
that the spores germinate like those of Tilletia Tritici. They figure one 
germinating spore and a small promycelium with sporidia at the end, 
not, however, attached to a spore. Clinton (1900) figures germinating 
spores of this fungus from redtop. He says the germ tube branched but 
did not form sporidia. The contents were mostly at the tip of the germ 
tube. A number of writers (Saccardo 1888, Plowright 1889, Schroeter 
1889, Brocq-Rousseu et Gain 1910, and Schellenberg 191 1) report that 
Fischer von Waldheim observed germination analogous to that of Tilletia 
Tritici. This impression has apparently arisen from his statement (Fischer 
von Waldheim, 1866), " Ciun Tilletia Carte sporarum evoluticiic congruit." 
As was pointed out by Oudemans (1893), this statement had reference 
to the production of spores in the mycelium and not to their germination, 
since later (1869-70:125) Fischer von Waldheim says: " Ungeachtet viel- 
fach wiederholter Versuche gelang es mir nicht die Sporen von Tilletia 
endophylla, de Baryana, .... zum Keimen zu bringen." 

In germination studies with this fungus the writer has used a consider- 
able number of substrata, among which may be mentioned the following: 
distilled water, tap water, Richard's full-nutrient solution^ (using potas- 
sium nitrate and ferric chloride in place of ammonium nitrate and ferrous 
sulfate, respectively), Cohn's modified solution,^ manure extract solution 
and agar,'^ soil extract solution and agar,^ hay infusion, extract from 
germinated timothy and redtop seedlings, extract from timothy and 
redtop flowers, moist filter paper, acetic acid solution 0.02 per cent, dilute 
solutions of ammonium hydroxide, ether, copper sulfate, calcium chloride, 
sulfuric acid, potassium permanganate. 

* The following methods were used in staining spores and germ tubes: The germinated sprres were 
transferred to slides coated with egg albumen. The drop or drops were allowed to concentrate as much 
as possible without drying, and two or three drops of fixer, usually Flemining's weaker solution, were 
added. After allowing this to concentrate, the slide was passed thru grades of alcohol up to ninety- 
five per cent, and after bringing back to a weaker alcohol or to water it was then stained with either Flem- 
ming's triple stain or Heidenhain's iron-haematoxylin. In some cases the spores were germinated directly 
on the slide coated with egg albumen and fixed without transferring. Occasionally the spores were germi- 
nated on a very thin film of agar on a glass slide. This film of agar, with the germinated spores, was then 
fixed and stained. However, the agar was so quickly covered by foreign organisms that the method was 
of little value. The writer has not succeeded in obtaining viable spores £ree from bacteria or other fungi. 

The material for examination of mycelium and spore formation was fixed in Flemming's weaker 
solution or in chromo-acetic acid solution. When the material was not too thick, no trouble was experienced 
in securing penetration of the fixing solution. For staining, Flemming's triple stain, Heidenhain's iron- 
haematoxylin, and Mayer's haemalum were used. As counter stains, orange G, eosin, and light green 
were employed either in aqueous solution or in clove oil. In some cases Heidenhain's iron^haematoxyliri 
and Mayer's haemalum were combined. . In this combination the iron-haematoxylin stains the nuclei 
while the haemalum stains the gelatinous sheath. 

'Richards, H. M. Jahrb. wiss. Bot. [PringsheimJ 30:667. 1897. 

6 Kellerman, W. A., and Swingle, W. T. Kansas Agr. E.xp. Sta. Rept. 3:229-231. 1890. 

'Jensen. C.N. Cornell Univ. Agr. Exp. Sta. Bui. 315:431-432. 1912. 

8 Jensen, C. N. Cornell Univ. Agr. Exp. Sta. Bui. 315:430-431. 1912. 



204 Bulletin 381 

Two methods for obtaining spore gennination were used. In the first, 
the spores were placed in drops of the solution on slides supported in petri 
dishes. To prevent evaporation, the bottom of each petri dish was 
covered with water or with some of the liquid to be tested. In the second 
method, the spores were allowed to dry on the cover glass and were then 
covered with a drop of agar, thus bringing the spores nearer the cover 
glass for examination. 

In the spring of 1914 the writer obtained a small percentage of germi- 
nation in a one-tenth-per-cent ether solution of spores taken from diseased 
timothy plants in the greenhouse. He has since made repeated attempts 
to germinate fresh spores both from these plants and from other timothy 
plants, but only an occasional spore has germinated. A considerable 
quantity of material was also collected, part of which was kept in the 
laboratory and part placed in wire netting outside. From time to time 
during the fall, winter, and succeeding summer, attempts were made to 
germinate these spores, but without success. A small percentage of 
germination has been obtained two or three times with spores from 
Kentucky bluegrass. 

Much better germination has been obtained with spores from redtop, 
in one instance over ninety per cent of the spores germinating. The 
proper conditions for spore germination have not been determined, but, 
as shown by the following observations, spores seem to retain their vitality 
longer if kept in a moist atmosphere. In the above-mentioned case 
of ninety per cent germination, the spores were taken from what appeared 
to be rather young son — that is, the epidermis was still intact or had 
just been ruptured. The plants had been brought into the laboratory 
and placed in a moist chamber above water. Spores taken from these 
plants twenty-four hours later showed about twenty per cent germination, 
and after forty-eight hours no further germination was observed. In 
another case plants were brought into the laboratory, and fresh spores 
taken from them and placed under favorable conditions germinated to 
the extent of fifteen per cent. Half the plants were placed in a moist 
chamber above water, while the others were left in the open laboratory. 
The next day spores from the plants in the moist chamber showed about 
four per cent germination, while all those taken from the plants left exposed 
failed to germinate. Similar results have later been obtained at different 
times. As will be shown later, the age of spores in a single sorus varies 
considerably, so that it is not possible to tell with certainty the age of 
spores that may germinate. The writer has never germinated any spores 
taken from sori that he knew to be very old. 

The manner or the abundance of spore germination does not seem 
to be affected by the medium in which the spores are placed. While 



Leaf Smut of Timothy 205 

the writer has found considerable variation in the germination of different 
lots of spores, this variation occurred more or less in all the media employed. 

The usual method of germination is for the germ tube to push out 
thru a hole that it makes in the spore wall. In some cases the wall 
cracks, due to the pressure exerted (fig. 54, o). Alter the contents of 
the spore have passed out, the crack is nearly closed. The germ tube 
continues to elongate, the contents of the spore becoming vacuolate 
(fig. 54, b-e). At about the time the germ tube is put forth, the large 
nucleus in the spore divides (fig. 54, a). Actual mitotic figures were not 
observed, but apparently four nuclei are produced in the spore before 
migration into the .tube. In figure 54, b and c, there are three nuclei in 
the tube with one still remaining in the spore. These nuclei are con- 
siderably smaller than the mother nucleus. They are usually more or 
less ellipsoidal and not over two microns in their longest diameter. Each 
has a single, rather large, deeply staining nucleolus located usually near 
the periphery of the nucleus. These nuclei pass out with the contents 
of the spore and are usually found grouped closely together in the germ 
tube (fig. 54, F-j). By the time the germ tube has reached a length of 
from fifty to one hundred microns, the entire content of the spore has 
passed into it, leaving a clear space behind (fig. 54, f, p, and fig. 55, a, b). 
The protoplasm at the end of the tube nearest the spore is usually much 
vacuolated (fig. 54, o, r, and fig. 55, a, b). With continued growth of 
the tube, the protoplasmic content, with the four nuclei, is found always 
at the growing tip (fig. 54, i-k, s, and fig. 55, a, b). From time to time 
hyaline cross-walls are laid down behind the protoplasm. These walls 
consist apparently of dried hyaloplasm. They originate at the rear of 
large vacuoles. 

In the majority of cases growth continues in this manner indefinitely, 
the protoplasmic content, with the nuclei, continuing at the tip. The germ 
tubes may pass out of the water or other medium and grow for a con- 
siderable distance across the slide. They seem to grow equally well 
whether immersed in the liquid or on the surface. In many cases side 
branches are pushed forth by the germ tube, the protoplasmic content 
filling both the tip and the side branches. In most of' these instances 
the protoplasm eventually withdraws from the side branch and continues 
in the tip, or withdraws from the tip and passes into the side branch 
(fig. 55, b). Occasionally the protoplasm becomes much vacuolated 
between the tip and the side branch, and later separates at one of the 
largest vacuoles, one half continuing in the tip and the other passing 
into the side branch (fig. 54, m, n, and fig. 55, a). In some cases the 
germ tubes become exceedingly branched, as shown in figure 54, l-n. 
The nuclear phenomena in these branched germ tubes were not studied. 



2o6 BUJ.fETJN 381 



Fig. 54. GERMINATION OF SPORES OF USTILAGO STRIAEFORMIS 

The spores were taken from redtop, with the exception of those in H, I, J, and O, which 
were taken from Kentucky bluegrass. The spores were germinated in either tap water or 
distilled water. 

A, Early stage of spore germination, showing the binucleate condition. Both the exo- 
spore and the endospore are visible. X 1250 

B-F, Later stages of germination, showing the passage of the nuclei and the protoplasmic 
contents into the germ tube. X 1250 

G-J, Late germination stages, showing the protoplasm and the nuclei in the tip of the 
germ tube. The nuclei remain grouped near together. In H and J the empty tubes at the 
base have collapsed in places and have stained dark. X 1250 

K, Germinated spore after 72 hours. X 325 .... 

L-N, Germinated spores, showing irregular branching of germ tubes and division of 
protoplasmic contents into two parts. X 350 

O, germinated spore, showing crack in the spore wall. X 735 

P-S, Various stages in the germination of a single spore. Drawings made after 10 hours, 
16 hours, 18 hours, and 48 hours, respectively. X 735 

T-W, Germinated spores, showing septa and clamp connections. Drawing from a cul- 
ture 48 hours old. X 735 



Leaf Smut of Timothy 



207 




Fig. 54. GERMINATION OF SPORES OF USTILAGO STRIAEFQRMIS 



208 



Bulletin 381 




Fig. 55. MYCELIUM AND SPORE GERMINATION OF USTILAGO STRIAEFORMIS 

A, Germination in distilled water of a spore from redtop, showing division of protoplasm into two 
parts. X 350 

B, Germination in distilled water of a spore from redtop, showing formation of side branches without 
division of protoplasm. X 350 

C, Intercellular mycelium in a timothy stem. The mycelium appears to pass thru the cells, but is 
merely applied closely to the cell walls. X 7IS 

D, Intracellular mycelium in base of a timothy stem. It is applied closely to the cell nucleus. X 7iS 
^, Intracellular mycelium in a vascular bundle of a timothy stem. X 71.$ 



Leaf Smut of Timothy -^og 

In one lot of spores collected on October 5, 19 14, a radically different 
method of germination was observed in the case of a few spores. These 
spores were placed in drops of water on slides in petri dishes. When 
examined again forty-eight hours later, the germ tubes or promycelia 
from a few spores on two of the slides were found to be septate, with 
well-developed clamp connections (fig. 54, u-w). In one case three cells 
were united by the clamp connections (fig. 54, t). One of the object 
slides was set aside to observe further development of the promycelia, 
while the spores on the other object slide were transferred to a slide coated 
with egg albumen and stained according to the method already described. 
Unfortunately none of the septate promycelia adhered to the slide. Further 
development of the promycelia on the slide set aside was apparently 
arrested by the strong light of the microscope, and the culture soon becarfie 
contaminated with yeasts and other organisms. These spores appeared 
in all respects like the normal spores found on redtop. The diseased 
plants were collected in a meadow and were wrapped in paper before being 
brought to the laboratory. The spores were then taken from the sori with 
a flamed scalpel, and therefore it was hardly possible that there was con- 
tamination of spores from any other species of Ustilago. This production 
of cross-walls adds weight to the contention that the fungus is a member 
of the genus Ustilago, even tho no conidial production was observed. 

Mycelhim 

The mycelium of the leaf smut fungus is especially distinguished by the 
formation of short side branches or knobs (fig. 55, c-e, and Plate xvii, i). 
The hyph^e are most frequently from 2 to 3 ^ in diameter, but may 
vary from 1.5 to 5 /x. The length of the cells varies from 4 to 30 /x. The 
mycelium is usuall}^ intercellular, in which case it sends out side branches 
which may penetrate the cells as haustoria or may merely apply them- 
selves closely to the walls of the host cells (fig. 55, c). In many cases, 
however, the mycelium is intracellular (fig. 55, d, e, and Plate xvir, i). 
A single mycelial thread growing through a cell and applied directly to 
the nucleus is shown in figure 55, d. 

The mycelium invades all parts of stem, leaves, and rhizomes, occa- 
sionally even penetrating the inner wall of the epidermal cells. In badly 
diseased plants the tissues are found very thoroly infested with the 
mycelium, in which case it may even grow into the vascular bundles 
(fig. 55, E, and Plate xvii, 2). With renewed growth of the plants in 
spring the myceHum follows the growing tip of the shoots, passing into 
the leaves as these are developed. In the leaves it -is usually found grow- 
ing alongside and parallel to the vascular bundles, or it may be found 
in the bundle itself. 



^i6 Bulletin 381 



Fig. 56. MYCELIUM AND SPORE FORMATION OF USTILAGO STRIAEFORMIS 

Drawings made from sections of stems or leaves of Dactylis glomerata 

A-L Mycelium in various stages. Binucleate stages are shown in B-E, H, and I. Four- 
nucleate stages are shown in A, F, and G. In G, H, and I the mycelium is shown with a gelat- 
inous wall, the beginning of spore formation. The cell shown at the left in H, which was at 
the edge of a sorus, had not yet begun to gelatinize. X 1670 

J-M, Short segments of the spore-forming threads, inostly binucleate. In most cases 
only the nucleolus can be made out with certainty at this stage. A gelatinous sheath was 
observed in only one case. X 1670 



Leaf Smut of Timothy 



211 




Fig. 56. MYCELIUM AND SPORE FORMATION OF USTILAGO STRIAEFORMIS 



212 Bulletin 381 

Considerable difficulty was experienced in staining nuclei and septa 
in the same mycelium. However, so far as observed, the cells of the 
vegetative mycehum are always binucleate (fig. 56, a-e. h, i). Division 
of the nuclei was not observed, but in a few cases a four-nucleate stage 
was found (fig. 56, a, f, g). This had apparently resulted from a more 
or less simultaneous division of the two nuclei, the septum not yet having 
been laid down. The nuclei are occasionally found side by side, but are 
usually at some distance apart in the cell. The point of origin of the 
binucleate condition was not determined, but in the case of the promycelia 
with clamp connections shown in figure 54, v and w, it is probable that 
the binucleate condition arose at this point. Whether this formation of 
septa and fusion of adjacent cells is a common occurrence before infection, 
the writer has no means of knowing at present. 

Many of the vegetative cells have clamp connections at the septa 
(fig. 56, B, c, f). These are formed as an outgrowth of one of the cells, 
apparently the terminal cell. A wall is laid down between this outgrowth 
and the parent cell. Whether the wall between this connection and the 
other hyphal cell is dissolved was not determined with certainty; but 
if it is, another wall is quickly laid down so that the clamp is cut off from 
both cells. So far as observed, the nuclei did not pass through this clamp. 
It has been suggested by Kniep^ that the clamp connection may serve 
for facilitating food transfer by exposing a larger surface for osmosis. 
If that is the case here, it is difficult to see why a wall should be laid down 
between it and both cells. 

Branching of the vegetative mycelium occurs at the septa (fig. 56, e, g). 
Such a branch, containing two nuclei with the septum not yet laid down, 
is shown in figure 56, g. 

Spore formation 

The only account in literature of the mycelium and spore formation of 
the leaf smut fungus is by Fischer von Waldheim (1869-70), who studied 
the fungus on Holcus mollis. He states that the spores are formed on 
the ends of threads, like those of Tilletia Caries, but, on the other hand, 
the threads are larger in circumference and a gelatinous membrane sur- 
rounds the spore until maturity, as in the typical species of Ustilago. 

Spore formation may occur in any region of the plant above ground. 
It usually originates in the parenchyma tissues of the leaf or in the cortical 
tissues of the stem outside the ring of sclerenchyma fibers. The mycelium 
that is to give rise to spore-forming threads begins to branch profusely 
in the tissues, producing a tangled mat of threads. This mycelium may 
remain intercellular for some time, forcing apart and crushing the cell 

'KniepHans. Zeitsch. Bot. 5:619. 1913. 



Leaf Smut of Timothy 213 

walls by its continued growth and branching. Eventually, however, it 
penetrates the cell and here continues its growth, branching profusely 
and absorbing the cell contents, the nucleus being the last thing to dis- 
appear. A change now appears in the mycelium. The wall begins to 
gelatinize and the lumen becomes narrower and more deeply staining 
(fig. 56, H, i). Meanwhile the mycelium breaks up into short cells, usually 
not over from five to twelve microns in length. The cells may be branched, 
resulting in a Y-shaped appearance; or, as frequently happens, they may 
be U-shaped, due to a bending-back of the myceliimi. In most cases 
these threads are densely intertwined and it is difficult to follow them 
for any distance (fig. 56, j, k). In rare cases, however, they grow out from 
the main sorus as septate, parallel strands (fig. 57, a). As the lumen 
grows narrower, it becomes increasingly difficult to stain the nuclei. 
In most cases only the nucleolus can be made out with certainty. As 
shown in figure 56, j-m, two nuclei are still usually present. Whether some 
cells are originally cut off with only one nucleus could not be made out 
with certainty. Meanwhile the gelatinous sheaths of the adjacent cells 
have become pressed together and apparently fused, so that it is impossible 
to distinguish them. Here and there individual cells soon begin to enlarge. 
It is during or just before this enlargement that nuclear fusion usually 
takes place. Only occasionally is a cell that has enlarged sufficiently 
to shov^ the nuclei found to have more than one nucleus. Two such 
immature spores, with two nuclei side b}^ side, are shown in figure 53, a. 
In another spore of figure 53, a, is shown a slightly later stage, in which 
the nucleus contains two nucleoli. 

The spore-forming threads are crowded so closely together in the 
young condition that it would be manifestly impossible for all the cells 
to produce mature spores without an enormous increase in the size of 
the sorus. Consequently it appears that many of the cells disintegrate 
(fig. 57, I, M, R, s). Whether some or all of these cells had only one 
nucleus at the beginning of spore formation it is impossible to say'. In 
the main body of the sorus the spore-forming threads and the young 
spores are so closely packed and interwined that their development cannot 
be followed accurately. In order to make out any details it is necessary 
to examine the isolated spores or threads around the border of the sorus. 
Here it is seen that the spores at the ends of the threads or the side branches 
are the first to be formed (fig. 57, g-j, o). Only very rarely is the 
maturest cell not at the end of the thread (fig. 57, f). However, a careful 
examination under favorable conditions shows that the cells farther back 
on the threads may eventually form spores also (fig. 57, e-h, k., n, o, r-t). 
In most cases this relation is very difficult to make out, due to the fact 
that the first-formed spore usually rounds up and loses all apparent 



214 Bulletin 381 



i 



Fig. 57. SPORE formation of ustilago striaeformis 

Drawings made from sections of stems or leaves of Dactylis glomerata. All X 1670 
A, Segments of spore-forming threads, with two immature spores 
B-D, Immature spores, showmg pointed ends 

E, Two immature spores attached end to end 

F, Three spores in a row, with the maturest one in the middle. Small, disintegrating 
masses of protoplasm are shown between the spores. The gelatinous sheath is only partly 
visible 

G, H, Terminal and intercalary spore formation, showing also a well-developed gelatinous 
sheath 

I, J, Terminal spore formation 

K-T, Terminal and intercalary spore formation. A spore is shown on a side branch 
in O. The production of spines is shown in R-T 



illl 



Leaf Smut of Timothy 



215 




Fig. 57. SPORE formation of ustilago striaeformis 



2i6 Bulletin 381 

connection with the other cells in the thread back of it. It is probably 
due to this fact that Fischer von Waldheim (1869-70:85) states: "Einer- 
seits bildet sie [ Tilletia de Baryana] ihre Sporen an den Enden der Faden, 
wie Till. Caries und endophylla." This intercalary formation of the 
spores in the spore-forming threads adds weight to the contention that 
the organism is a species of Ustilago rather than of Tilletia. In only 
one instance has the writer established the connection of more than two 
spores in a thread (fig. 57, t). In that instance the spores were formed 
in a thread which had extended considerably beyond the end of the sorus 
and had plenty of room and nutritive material in which to develop. 
The spores are seen to be older and to have larger spines at the upper 
end, while they become progressively younger toward the bottom., which 
was the point of connection with other threads. 

If stained under favorable conditions, the young spore is found always 
to have a gelatinous sheath surrounding it (fig. 57, g-t). In its early 
stages the spore is usually more or less pointed at one or both ends (fig. 
53, A, B, and fig. 57, b-i). In some cases these ends are blunt, in others 
they are long and sharp. As the spore enlarges the ends become rounded 
and the gelatinous sheath is pushed out. There is no visible wall about 
the spore, other than that formed by the gelatinous sheath, until it 
is nearly two-thirds grown. About this time, however, the spore becomes 
set off from its sheath by a thin wall, on the outside of which appear 
small granules which are the beginnings of the spines (fig. 53, c). As 
the spore matures the wall becomes darker and the spines become 
longer and thicker. The growth of the spines appears to be due partly 
to drying and shrinking of the material in the interstices, and partly to 
outward growth of the spines themselves. The relation of the spines 
to the spore wall is most clearly shown by plasmolyzing the contents 
slightly. By the time the spores are mature, the gelatinous sheath has 
entirely disappeared (fig. 53, d, e). 

In the young sorus the first spores are formed in the center. As the 
sorus becomes older, spore formation gradually proceeds outward (Plate 
XVII, 3). In some cases the mycelium spreads no farther than the limits 
it occupied when spore formation began; but in the majority of cases 
it continues to invade new cells, branching and giving- rise to additional 
spore-forming threads. It is due to this continued progress of the mycelitim 
that fusion of adjacent sori occurs. 

Inoculation and infection 

No inoculation experiments with this fungus have been reported in 
literature. Clinton (1900) says that infection probably occurs thru the 
germinating seed, but he cites no experimental work. From experi- 
ments of the writer it appears that inoculation and infection occur at 



Leaf Smut of Timothy 



217 



blossoming time. The spores are carried to the opening flowers either 
by wind or by insects. Here they germinate, sending out a germ tube 
which penetrates into the ovary and remains in the young embryo in 
a more or less dormant condition until it begins growth after planting: 
Seed inoculation. — On November 4, 19 13, timothy seed bought of a 
local dealer was inoculated with spores of Ustilago striaeformis taken 
from timothy plants that had been kept in the laboratory for three months. 
Part of this treated seed was sown in the greenhouse along with clean 
seed. The remaining treated seed was sown in a box, and after germi- 
nation had started the box was kept in a rather cool room until the plants 
were between two and three inches high. These plants were then placed 
in the greenhouse. The disease made its first appearance on the leaves 
of a number of the plants about four months later, and on April i, 19 14, 
the results shown in table i were obtained : 

TABLE I. Results of Timothy Seed Inoculations Made on November 4, 1913 



Percentage of smutted 
plants 



Inoculated 



Check 



Plants kept in greenhouse. . . . 
Plants first kept in cool room. 



2.0 
1-9 



2.5 
1.8 



After April i only one additional plant became diseased. Some of these 
plants later became so badly diseased that they died, while a few of the 
others produced seed on one or more shoots of the stool. 

On April 18, 19 14, a series of inoculations were made on thirty-two 
species of grasses, using a mixture of fresh spores from timothy, spores 
that had been kept outside over winter, and spores that had been kept 
in the laboratory for several months. The seed was inoculated by mixing 
it with smut spores in water. Timothy seeds from five different sources 
were used, redtop seeds from three sources, and Kentucky bluegrass 
seeds from three sources. On July 22, when these plantlets were examined, 
those of the timothy from two sources showed a small percentage of 
smutted plants in the case of both treated and untreated seeds (table 2). 
All the other plants remained healthy. 

TABLE 2. Results of Timothy Seed Inoculations Made on April 18, 1914 





Lot I 


Lot 2 




Number 
of stools 


Percentage 
smutted 


Number 
of stools 


Percentage 
smutted 


Treated 


146 
206 


2.0 


291 
217 


1 .0 


Check 


19 


14 



2i8 Bulletin 381 

On May 11, 19 14, a series of inoculations similar to those described 
above were made, using eight species of grasses, including seeds from 
two sources each of redtop, Kentucky bluegrass, and timothy. When 
the plantlets were examined on July 22, timothy plants from one source 
(lot I of table 2) showed a small percentage of diseased plants in the 
case of both treated and untreated seeds (table 3). When examined 
again on August 1 7 the number of diseased plants in this lot had increased 
slightly, but all the other plants were healthy. 

TABLE 3. Results of Timothy Seed Inoculations Made on May ii, 1914 



Number 
of stools 



Percentage of smutted 
plants 



July 22 August 17 



Treated . 
Check. . 



197 
144 



••5 



2.5 



2.1 2. J 



As shown in the tables, the number of smutted plants in these experi- 
ments was in no way affected by inoculating the seed.' The experiments 
are inconclusive, however, since the spores failed to germinate in con- 
temporaneous germination tests. 

Blossom inoculation. — Blossoms of redtop, orchard grass, timothy, 
and Kentucky bluegrass were inoculated with spores taken from each of 
the hosts. The inoculations were made in most cases either by dusting 
the spores on the stigma or by spraying them on in water with an atom- 
izer. Unfortunately the plants used in this experiment were later acci- 
dentally cut down, thus destroying the experiment. 

Later in the summer these inoculations were repeated on second-growth 
timothy blossoms, using spores from timothy and redtop. A number of the 
resulting seeds were placed to germinate between moist filter papers, 
and as soon as growth started sufficiently to show that the seeds were 
not killed they were fixed and infiltrated with paraffin, and sectioned. 
In one case typical smut mycelium was found in the seed, thus showing 
that infection had occurred. The remaining seed was sown in the green- 
house and later transplanted to the field, or was sown directly in the 
field, but owing to the extremely wet season the plants were completely 
smothered by weeds during the writer's absence. The writer expects 
to repeat these experiments on a more extensive scale. 

In the summer of 191 4 a quantity of viable timothy seed was collected 
from diseased plants. Some of this seed was germinated between moist 
filter paper, and as soon as sufficient growth had started to be sure that 



Leaf Smut of Timothy 



219 



the seeds had not been killed by the fungus they were infiltrated with 
paraffin and sectioned. In a few of these mycelium was found (fig. 58). 
It was not possible, however, to tell whether this mycelium had come 
from blossom infection or had grown into the seed through the funiculus 
from the diseased rhachilla. The remaining seed from these plants was 
sown in the field, but suffered the same fate as that from the blossom 
inoculation experiment mentioned above. 

Soil inoculation. — In order to test the possi- 
bility that the spores might live in the soil for 
some time, the following experiment was per- 
formed: On April 14, 19 14, plots 2, 3, 
4, and 5 (fig. 59) were inoculated with 
fresh spores from timothy. On the same 
day timothy seed procured from 
a local dealer was sown in plots 
I and 5, and seed treated by 
covering it with 
spores was sown in 
plot 6. After one 
week seed was sown 
in plots 4 and 7, 
after three weeks 
in plot 2, and after 
six weeks in plot 3 . 
No diseased plants 
were produced in 
any of the plots. 
This experiment is 
inconclusive, since 
no germination of 
spores was ob- 
tained in contem- 




FiG. 58. 



SECTION THROUGH A TIMOTHY SEED, SHOWING MYCE- 
LIUM IN THE EMBRYO. X I 75 



poraneous germi- 
nation tests. 

Inoculation of growing tissues. — On March i. 19 14, eight timothy 
plants were inoculated with both fresh and old spores taken from dis- 
eased timothy. In some cases the spores were placed on the uninjured 
growing tissues at the top, while in others the tissues were injured by 
needle pricks or by cutting with a scalpel. In some of the stools a 
number of the stalks were cut off and the young sprouts that started 
out were covered with spores. In all cases the plants were kept moist 



220 



Bulletin 381 



by covering them with a bell glass. No infection was obtained on any 
of the plants. 

Later in the summer these experiments were twice repeated on timothy 
and redtop, using spores from timothy, redtop, and bluegrass. The spores 
from redtop showed from five to twelve per cent germination on slides 
in petri dishes. In no case did any infection result. 

Examination of inoculated seedlings. — Timothy and redtop seeds were 
inoculated with spores from timothy and redtop, respectively, and placed 
in a moist chamber between moist filter papers. The spores from redtop 
showed about ten per cent germination. Two days later additional spores 
were dusted on the seeds, and in this case the spores from redtop showed 
four per cent germination. The germinated seedlings were removed 



I 


2 


3 




Check 


3 weeks 


6 weeks 


7 
Check 








4 


5 


6 




I week 


Seed sown 


Seed 






at once 


inoculated 





Fig. 59. CHART SHOWING PLAN OF SOIL INOCULATION EXPERIMENT 



from time to time and were fixed and sectioned in paraffin, but in no 
case was any mycelium found in the tissues. 

Summary of life history 

The fungus may pass the winter in three different ways: first, as 
mycelium and spores in the green tissues of the plants ; second, as mycelium 
in the dormant embryo of the seed; and third, as mycelium in bulbs and 
rootstocks of perennial plants. In the first method the fungus persists 
in the green tissues, usually spreading very little if at all until renewed 
growth starts in the spring. However, over the steam pipes on the Cornell 
University campus, where the grass maintains a slight growth during 
the winter, the writer has found the fungus active thru the entire winter. 
In the second case, when the seed is sown the mycelium becomes active, 
growing up with the young plant and spreading out into the leaves, where, 
after a period varying from two and one-half to six months or more, it 
first makes itself evident by the elongate sori. In the third case, when 



Leaf Smut of Timothy 22 r 

the plants start growth in the spring the mycehum grows out into or 
with the new shoots, producing the lead-colored sori very soon after 
growth starts. Additional sori are produced all summer whenever there 
is any new growth of the diseased plants. In those plants that produce 
underground stems the mycelium grows thru these, keeping pace with 
the growing tip and establishing itself in the newly formed plants. The 
writer has found plants of Kentucky bluegrass and a creeping variety 
of AgrosHs alba affected in this manner at a distance of over four feet 
from the parent plant. At blossoming time the spores are distributed 
to the stigmas of the opening flowers, where they germinate, giving rise 
to a germ tube which penetrates into the ovary, in this way infecting 
the seed. When a plant is once infected, it apparently never becomes 
free from the fungus. The writer has observed the disease in the same 
plant for three successive seasons. The old dead leaves, showing sori 
formed the previous summer, may be found in the spring surrounding 
the new shoots, which soon show the disease. 

PATHOLOGICAL HISTOLOGY 

The only mention in literature of the effects of this fungus on the tissues 
is by Strohmeyer (1896). He gives a brief account of the alterations 
caused on a number of different plants. 

An examination of diseased leaves shows that the sori originate in the 
mesophyll between the vascular bundles (Plate xvii, 5 and 6). They may 
originate either near the upper or the lower epidermis, or midway between 
them. The mesophyll cells surrounding the young sorus are frequently 
found to have increased in diameter and to have lost their chlorophyll 
content. The cell walls persist for some time after the contents of the 
cells have been absorbed, and may be found extending into the young 
sorus as isolated strands. Eventually, however, they disappear. If the 
sorus originates near the surface of the leaf, the epidermal cells are early 
found to have increased in diameter, especially tangentially, apparently 
even before any particular pressure is exerted on them by the enlarging 
sorus, since they may be hypertrophied for a considerable distance above 
or below the sorus. As the sorus increases in size, additional mesophyll 
cells are invaded and are broken down partly by pressure and partly by 
dissolution of the walls. At the same time the epidermis is pushed out 
due to this pressure, the cells increasing greatly in tangential diameter 
and becoming somewhat flattened (Plate xvii, 6). 

In the case of large sori the vascular bundles on either side are forced 
to one side and the nourishing cells surrounding them are crushed. In 
most cases the xylem and the phloem elements appear to be very little 
affected. The walls of the sclerenchyma fibers accompanying the larger 



222 Bulletin 381 

bundles are frequently less lignified than those above or below the sorus. 
Adjacent sori occasionally may fuse laterally, in which case the bundle 
between them is pushed toward one epidermis, usually the upper, while 
the opposite epidermis is pushed out. When a large sorus is formed 
adjacent to a small vascular bundle consisting of only three or four cells, 
the bundle may be completely obliterated at that point but will still 
be found above and below the spore mass. Cross-connections of the 
bundles may be either pushed aside or completely destroyed. 

The spore-forming mycelium continues to spread until it reaches one 
or both of the epidermal layers. The uninjured epidermal cells are very 
seldom invaded by the mycelium. As the sorus becomes larger the 
epidermal cells may be crushed and ruptured by the pressure from within, 
or, as frequently occurs, the inner wall of the epidermal cells is dissolved 
by the fungus, the spores then pressing against the outer wall. This 
is later ruptured either by pressure or by the solvent action of the fungus. 
The writer has found both the lower and the upper epidermis to be 
ruptured in diseased leaves kept undisturbed under a bell glass. In 
this case the rupture of the second epidermis could have occurred only 
by the wall being dissolved until it became exceedingly weak. 

In the stem the sori are usually found in the cortex just beneath the 
epidermis and outside the ring of sclerenchyma fibers (Plate xvii, 4). 
The cortical cells are broken down and the epidermal cells are enlarged 
in their tangential diameter and pushed out. The epidermal cells are 
eventually ruptured just as they are in the leaves. 

EFFECT OF ENVIRONMENTAL FACTORS 

Ule (1884:216) reports that protected places, especially where protected 
in winter by snow as on the west side of hills, are favorite places for this 
and related fungi. He rarely, if ever, found the disease on open meadows. 
Griffiths (1903) states that in CaHfornia the disease seems to be confined 
to well-drained areas abundantly supplied with seepage from ditches, 
rather than to poorly drained or drier parts of meadows. 

In New York the writer has not observed any difference in the amount 
of this disease between wet and dry soils or exposed and protected places, 
provided the grass was pastured or otherwise kept to the same size in 
both locations. However, especially in the case of Kentucky bluegrass, 
if the plants are allowed to reach maturity there is usually much less 
smut in the rich, moist soils. This is apparently due to the fact that 
in the rich soils the healthy plants grow so rank and tall that they are 
able to crowd out the diseased, stunted plants. This probably accounts 
for the fact that few diseased Kentucky bluegrass plants can be found 
along moist roadsides, while they are extremely common on lawns where 
the grass is mowed. 



Leaf Smut of Timothy 



223 



CONTROL 

No experiments are recorded in literature on the control of leaf smut, 
but Pammel (1890) and Clinton (1900) have suggested the possibility 
of controlling the disease by seed treatment. 

Since, as already shown, infection occurs thru the blossoms, it follows 
that if the grower plants seed free from the smut fungus, the grass will 
be entirely free from the disease. This method, however, is not feasible 
under most conditions, since the disease is so universally present. 
Further, many growers buy their seed from dealers and thus have no 
means of knowing where the seed came from or what percentage of it 
may be infected. In such a case the only remedy lies in treating the seed. 

EFFECT OF SEED TREATMENT ON GERMINATION 

Before any experiments were undertaken on the control of this disease 
by seed treatment, a nimiber of germination tests with timothy seed were 
performed in order to determine the point of injury to the seed by the 
various treatments. The seeds were treated and then germinated between 



TABLE 4. 



Effect on Germination of Timothy Seed, of Treatment with 
Formaldehyde and Copper Sulfate Solutions 



Treatment 


Percentage of germination 


Lot I 


Lot 2 


Control, soaked in water i minute 

Control, soaked in water i hour 

Control, soaked in water 2 hours 


97 
95 
92 
92 


60 

64 


Control, soaked in water 10 hours 


64 




Formaldehyde solution, 40 per cent, i pint to 38 gallons 
of water 

§ hour 


95 
94 
94 
91 
90 

85 




I hour 




2 hours 




4 hours 




10 hours 




24 hours 








Formaldehyde solution, 40 per cent, i pint to 76 gallons 
of water 

5 hour 


94 
92 

93 
90 
86 

82 


53 
55 
SI 


I hour 


2 hours 


4 hours 


51 
51 


1 hours 


24 hours 


50 






Copper sulfate solution, 2 per cent 

I minute 


92 
88 
90 
91 


S4 


2 minutes . 


56 
54 
50 


5 minutes 

1 minutes 



224 



Bulletin 381 



moist filter papers in petri dishes. The experiments were run in triplicate 
in each case, two hundred seeds being placed in each petri dish. The 
seeds in one petri dish were placed to germinate at once, while those in 
the other two dishes were first kept dry for forty-eight hours. It was 
found that in all treatments, including the checks, better germination 
occurred where the seed was placed to germinate at once after treating 
than where it was dried for two days. This increase amounted to from 
one to seven per cent. The averages for all three petri dishes are given 
in tables 4 and 5. The germination of seed after treatment with various 
formaldehyde and copper sulfate solutions is shown in table 4. From 
these results it is apparent that timothy seed may be treated with one 
pint of forty-per-cent formaldehyde solution to thirty-eight gallons of 
water for from two to four, or even ten hours, or with two-per-cent copper 
sulfate solution for ten minutes, without materially afi^ecting its germinating 
power. The results of treating timothy seed with hot water are given 
in table 5. Before the seed was plunged into hot water it was held for 
one minute in water at a temperature four or five degrees below that 
at which it was to be treated. The temperature of the water did not 
vary over 0.25 degree above or below the stated temperature, and in 
most cases not over 0.15 degree. Judging from the results given in table 5, 
favorable treatments would appear to be with water at 54° C. for ten 
minutes or 52° C. for fifteen minutes, with a previous soaking in cold 
water of from six to eight hours. 



TABLE 5. 



Effect on Germination of Timothy Seed, of Various Treatments 
WITH Hot Water 



Time 

soaked in 

cold water 

(hours) 


Time 

held in 

hot water 

(minutes) 


Temperature 

of hot 

water 
(centigrade) 


Percentage of germination 


Lot I 


Lot 2 


A 


Control 
Control 
Control 




92 
94 
95 




6 




60 


10 












4. 


5 
10 

15 
20 

25 

5 
10 

15 
20 

25 

5 
10 

15 
20 

25 


50° 
50° 
50° 
50° 
50° 

50° 

50° 
50° 
50° 

50° 
50° 


97 
96 
96 
95 
95 
95 
95 
95 
93 
92 

94 
90 

93 
94 

89 




4. 




4. 




A . . . 




4 

6 






6 




6 




6 




6 




10 




10 




10 




10 




JO 





Leaf Smut of Timothy 

TABLE 5 {concluded) 




225 


Time 

soaked in 

cold water 

(hours) 


Time 

held in 

hot water 

(minutes) 


Temperature 

of hot 

water 
(centigrade) 


1 
Percentage of germination 


Lot I 


Lot 2 


4 


5 

ID 

15 

20 
25 

5 

10 

15 

20 

25 

5 

ID 

15 

20 

25 


52° 
52° 
52° 
52° 
52° 
52° 
52° 
52° 

52° 
52° 
52° 
52° 
52° 
52° 


95 

92 

93 

92 
92 

97 
95 
94 
91 
89 

95 
95 
91 
89 

88 




4 




4 




4 




4 




6 


57 
45 
43 
33 
29 


6 


6 

6 

6 


lO 


ID 




10 




10 




lO 








4 


5 

lO 

15 

20 
25 

5 

ID 

15 

20 
25 

5 

ID 

15 

20 

25 


54° 

54 

54° 

54° 

54° 

54° 

54° 

54° 

54° 

54° 

K 

54° 
54° 
54° 


93 
93 
89 
89 
88 

94 
92 

85 
85 
83 

93 
88 

85 
86 

79 




4 




4 




4 




4 




6 


47 
44 
38 
27 
21 


6 


6 


6 


6 


ID 




ID . 




lO 




lO 




10 









EFFECT OF SEED TREATiMENT ON PERCENTAGE OF SMUT 

During the summer of 19 14 a number of experiments were conducted 
on the control of leaf smut by seed treatment. In one experiment the 
seeds, except those in a part of the check, were dusted with a mixture of 
spores taken from fresh plants and from dried plants kept over winter 
in the laboratory or exposed outdoors over winter. The results are 
given in table 6. As shown in the table, the dusting of spores on the 
seed had no effect on the amount of smut produced. However, the seed 
was already infected, as shown by the checks, so that data on seed treat- 
ment were obtained. The hot water treatments gave perfect control 
in both cases. The plots treated with formaldehyde and copper sulfate 
solutions showed less smut than the checks, buc, owing to the small number 
of plants used and the low percentage of smut, this may have been du^ 



226 Bulletin 381 

TABLE 6. Results of Treating Timothy Seed for Smut 



Treatment 



Check, no treatment 

Check, seed dusted with spores 

Formaldehyde solution, 40 per cent, i pint to 45 gallons 

of water for two hours 

Copper sulfate solution, 2 per cent, for two minutes 

Cold water for six hours, hot water at 52° C. for fifteen 

minutes 

Cold water for six hours, hot water at 54° C. for ten 

minutes 



Number 
of stools 



396 

444 

416 
406 

510 

322 



Percentage 
of smut 



2 
2.25 

15 
I 

o 

o 



to experimental error. The timothy seed was the same as lot i in table 
2. In the other experiments no smut occurred even in the check plots. 
Further experiments during the summer of 191 5 were nullified by wet 
weather. 

These results, while not conclusive, point strongly to the probability 
of controlling this disease by treating the seed with hot water. 

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